Tumor Samples Research Articles

Atezolizumab Plus Chemotherapy With or Without Bevacizumab in Advanced Biliary Tract Cancer: Clinical and Biomarker Data From the Randomized Phase II IMbrave151 Trial.

Biliary tract cancers (BTCs) harbor an immunosuppressed tumor microenvironment and respond poorly to PD-1/PD-L1 inhibitors. Bevacizumab (anti-vascular endothelial growth factor) plus chemotherapy can promote anticancer immunity, augmenting response to PD-L1 inhibition. This randomized, double-blind, proof-of-concept phase II study enrolled patients (n = 162) with previously untreated advanced BTC (IMbrave151; ClinicalTrials.gov identifier: NCT04677504). Patients were randomly assigned 1:1 to receive cycles of atezolizumab (1,200 mg) plus bevacizumab (15 mg/kg) or atezolizumab plus placebo once every 3 weeks until disease progression or unacceptable toxicity. All patients received cisplatin (25 mg/m2) plus gemcitabine (1,000 mg/m2; cisplatin plus gemcitabine [CisGem]) on days 1 and 8 once every 3 weeks for up to eight cycles. Stratification of patients was by disease status, geographic region, and primary tumor location. The primary end point was progression-free survival (PFS). No formal hypothesis testing was performed. Exploratory correlative biomarker analysis was undertaken using transcriptome analysis (n = 95) and mutation profiling (n = 102) on baseline tumor samples. Between February and September 2021, 162 patients were enrolled. Median PFS was 8.3 months in the bevacizumab arm and 7.9 months in the placebo arm (stratified hazard ratio [HR], 0.67 [95% CI, 0.46 to 0.95]). Median overall survival (OS) was 14.9 and 14.6 months in the bevacizumab and placebo arms, respectively (stratified HR, 0.97 [95% CI, 0.64 to 1.47]). The incidence of grade 3 or 4 adverse events was 74% in both arms. High VEGFA gene expression was associated with improved PFS (HR, 0.44 [95% CI, 0.23 to 0.83]) in the bevacizumab arm versus placebo. In unselected patients with advanced BTC, adding bevacizumab to atezolizumab plus CisGem modestly improves PFS but not OS. High VEGFA gene expression may represent a predictive biomarker of benefit from atezolizumab/bevacizumab, warranting further investigation.

Neutrophil-related Signature Characterizes Immune Landscape and Predicts Prognosis of Invasive Breast Cancer.

As a leading prevalent malignancy, breast cancer remains a significant worldwide health issue. Recent research indicates that neutrophils play a crucial role in breast cancer development. The prognostic significance of neutrophil-related genes (NRGs) or the immune landscape of the neutrophil-related signature in invasive breast cancer (IBC) is, nevertheless, unknown. To uncover innovative therapy alternatives, the significance of the neutrophil-related signatures in IBC was evaluated here. Briefly, a prediction model based on neutrophil-related core prognostic genes and The Cancer Genome Atlas data was created (TCGA). The model may assess IBC patients' prognosis. The IBC data from the Gene Expression Omnibus (GEO) confirmed the prognostic accuracy of the model. The overall survival (OS) of patients was worse in the group with a high NRGs score compared to the group with a low NRGs score. In addition, patients with low NRGs scores were considerably more sensitive to vinorelbine, cyclophosphamide, epirubicin, gemcitabine, paclitaxel, 5-fluorouracil, docetaxel, and cisplatin. Patients with low NRGs scores responded better to CTLA-4 and PD-1 treatments. Additionally, the immune microenvironment components were more abundant in patients with low NRGs scores. Moreover, qRT-PCR results confirmed that LEF1 had a higher expression level in tumor samples compared to normal samples, whereas NRG1 and STX11 exhibited lower expression levels in tumor samples than in normal samples. These results suggest that NRGs might be utilized as biomarkers to predict the prognosis of individuals with IBC, thereby paving the way for the creation of customized therapies for IBC.

Characterization of Fibroblast Growth Factor Receptor Alterations and Activation in Patients with High-Risk Non-Muscle Invasive Bladder Cancer.

The GARNER study investigated fibroblast growth factor receptor (FGFR) alteration (ALT) frequency and the clinical outcome relationship with Bacillus Calmette-Guérin (BCG) treatment in high-risk non-muscle invasive bladder cancer (HR-NMIBC). An FGFR predictive response signature (FGFR-PRS) was discovered that identifies patients with an activated FGFR pathway who could potentially benefit from FGFR-targeted therapy beyond those who are FGFR ALT (+). Pre-treatment tumor samples and clinical data were analyzed from 582 BCG-treated HR-NMIBC patients. FGFR-PRS was discovered using a separate BC dataset and applied to the GARNER and other BC cohorts. FGFR-PRS was also applied to in vitro data from UC cell lines treated with FGFR-active agents. 31% of pre-treatment GARNER HR-NMIBC tumors were FGFR ALT (+) but this was not significantly associated with BCG response. For the subset of patients with paired pre- and post-BCG treatment samples, nearly one-third pre-treatment ALT (+) patients were ALT (-) post-treatment. FGFR-PRS identified patients with an activated FGFR pathway and identified approximately 2-fold additional patients compared to ALT status alone, and this increase was similar across tumor stage. A positive relationship between tumor growth inhibition and FGFR-PRS score was shown in BC in vitro models treated with FGFR-active agents. These data provide support for FGFR-targeted therapy use in FGFR ALT (+) HR-NMIBC and describe tumors with shared FGFR pathway-activated biology that are FGFR ALT (-) but FGFR-PRS (+). The latter suggests a broader potential patient population for FGFR-targeted therapy, which will require subsequent validation in patients treated with FGFR-targeted therapy.

Large Scale Comparative Analysis of Canine and Human Osteosarcomas Uncovers Conserved Clinically Relevant Tumor Microenvironment Subtypes.

Osteosarcoma is an aggressive bone cancer lacking robust biomarkers for personalized treatment. Despite its scarcity in humans, it is relatively common in adult pet dogs. This study aimed to analyze clinically annotated bulk tumor transcriptomic datasets of canine and human osteosarcoma patients to identify potentially conserved patterns of disease progression. Bulk transcriptomic data from 245 pet dogs with treatment-naïve appendicular osteosarcoma were analyzed using deconvolution to characterize the tumor microenvironment (TME). The TME of both primary and metastatic tumors derived from the same dog was compared, and its impact on canine survival was assessed. A machine learning model was developed to classify the TME based on its inferred composition using canine tumor data. This model was applied to 8 independent human osteosarcoma datasets to assess its generalizability and prognostic value. This study found three distinct TME subtypes of canine osteosarcoma based on cell type composition of bulk tumor samples: Immune Enriched (IE), Immune Enriched Dense Extra-Cellular Matrix-like (IE-ECM), and Immune Desert (ID). These three TME-based subtypes of canine osteosarcomas were conserved in humans and could predict progression-free survival outcomes of human patients, independent of conventional prognostic factors such as percent tumor necrosis post standard of care chemotherapy treatment and disease stage at diagnosis. These findings demonstrate the potential of leveraging data from naturally occurring cancers in canines to model the complexity of the human osteosarcoma TME, offering a promising avenue for the discovery of novel biomarkers and developing more effective precision oncology treatments.

HAS THE INITIATION OF A FORMAL PATHWAY FOR TUMOUR TISSUE COLLECTION IN NEURO ONCOLOGY PATIENTS LED TO AN INCREASE IN PATIENT’S HAVING WHOLE GENOME SEQUENCING (WGS), AND HOW WILL THIS IMPACT ONCOLOGY MANAGEMENT IN THE FUTURE?

Abstract AIMS To change the standard of care for collection of glial tumour tissue at a London teaching hospital. This would enable WGS on the tumour tissue, and give patients access to further clinical trials. METHOD The previous standard of care was for brain tumour pathology samples to be stored in paraffin. This was not suitable for WGS or for many clinical trials; the tumour samples needed to be sent to the lab as fresh tissue. The introduction of a genomics clinical nurse specialist meant that relationships were built between the surgical team, pathology teams and ward teams. There was a teaching by one of the neurosurgical team to the theatre staff. Genomics CNS led training for the nursing team. Adults started having WGS in May 2023 but many patient’s still had tissue incorrectly stored and could not have the sequencing performed. There was an introduction of a formal pathway in theatre created by one of the neurosurgeons in Dec 2023. RESULTS From May 2023 - November 2023, 58% adult glioma patients had WGS. Since December 2023 - current (March 2023) and the introduction of the formal pathways, 71% had WGS. CONCLUSION A formal pathway has now changed the standard of care for collecting tissue. There is still not 100% compliance, and this remains multifactorial. However the introduction of a formal pathway has now increased the percentage of eligible patients receiving WGS. This positive outcome has now led to discussions about freezing the fresh glioma tissue to allow glioma patient’s wider access to clinical trials which they would previously not have had access to.

Accurate identification of locally aneuploid cells by incorporating cytogenetic information in single cell data analysis

Single-cell RNA sequencing is a powerful tool to investigate the cellular makeup of tumor samples. However, due to the sparse data and the complex tumor microenvironment, it can be challenging to identify neoplastic cells that play important roles in tumor growth and disease progression. This is especially relevant for blood cancers, where neoplastic cells may be highly similar to normal cells. To address this challenge, we have developed partCNV and partCNVH, two methods for rapid and accurate detection of aneuploid cells with local copy number deletion or amplification. PartCNV uses an expectation-maximization (EM) algorithm with mixtures of Poisson distributions and incorporates cytogenetic information to guide the classification. PartCNVH further improves partCNV by integrating a hidden Markov model for feature selection. We have thoroughly evaluated the performance of partCNV and partCNVH through simulation studies and real data analysis using three scRNA-seq datasets from blood cancer patients. Our results show that partCNV and partCNVH have favorable accuracy and provide more interpretable results compared to existing methods. In the real data analysis, we have identified multiple biological processes involved in the oncogenesis of myelodysplastic syndromes and acute myeloid leukemia.

CELL-INTRINSIC METABOLIC PHENOTYPES IDENTIfiED IN GLIOBLASTOMA PATIENTS USING MASS SPECTROMETRY IMAGING OF 13C-LABELED GLUCOSE METABOLISM

Abstract AIMS Glioblastoma (GB) is an inherently heterogenous and invasive primary brain tumour. Genetic and transcriptomic studies have attempted to classify GB into subtypes that can identify therapeutic vulnerabilities and predict survival. An outstanding question and technical challenge is to visualise the metabolism of a tumour within its native microenvironment and understand to what extent its metabolism is driven by the microenvironment. METHOD Patients with GB tumours were infused with [U-13C]glucose and intra-operative multi-regional tumour samples were rapidly snap frozen for metabolic tracing analysis using mass spectrometry imaging (MSI). Tissue microenvironment was assessed using multiplexing of antibodies on immunocytochemistry (IMC) of parallel sections. Isotope tracing in human derived neurospheres and patient derived xenografts were used to confirm the presence of metabolic phenotypes detected in the human samples and to test susceptibility of each phenotype to a panel of drugs targeting cellular metabolism. RESULTS Three metabolic signatures were identified in patient GB tumours: glycolytic, oxidative and a mixed glycolytic/oxidative phenotype. The phenotypes did not correlate with microenvironmental features, including proliferation rate, immune cell infiltration, and vascularisation. The phenotypes were retained when patient derived cells were grown in vitro, or as orthotopically implanted xenografts, and were robust to changes in oxygen concentration, demonstrating their cell intrinsic nature. The spatial extent of the regions occupied by cells displaying these distinct metabolic phenotypes are large enough to be detected using clinically applicable metabolic imaging techniques. The phenotypes also demonstrate differential drug sensitivities. CONCLUSION This is the first report to demonstrate high resolution imaging of metabolic fluxes in a human tumour in vivo. In conjunction with studies on patient-derived neurospheres and orthotopically implanted xenografts we have demonstrated the presence of different metabolic phenotypes within GB that are tumour cell intrinsic and largely independent of the tumour microenvironment. These can be imaged clinically and used to stratify patients for personalised therapy.

Expression and its clinical significance of cell-cycle dependent kinase 1 in malignant peripheral nerve sheath tumors

To explore the role and clinical significance of cell-cycle dependent kinase 1 (CDK1) and its upstream and downstream molecules in the development of malignant peripheral nerve sheath tumor (MPNST) through the analysis of clinical tissue samples. A total of 56 tumor samples from MPNST patients ("Tianjin" dataset) who underwent surgical resection, confirmed by histology and pathology between September 2011 and March 2020, along with 17 normal tissue samples, were selected as the research subjects. MPNST-related hub genes were identified through transcriptome sequencing, bioinformatics analysis, immunohistochemistry staining, and survival analysis, and their expression levels and prognostic associations were analyzed. Transcriptome sequencing and bioinformatics analysis revealed that upregulated genes in MPNST were predominantly enriched in cell cycle-related pathways, with CDK1 occupying a central position among all differentially expressed genes. Further differential analysis demonstrated that CDK1 mRNA expression in sarcoma tissues was significantly higher than in normal tissues [based on searching the cancer genome atlas (TCGA) dataset, P<0.05]. In MPNST tissues, CDK1 mRNA expression was not only significantly higher than in normal tissues (based on Tianjin, GSE141438 datasets, P<0.05), but also significantly higher than in neurofibromatosis (NF) and plexiform neurofibromas (PNF) (based on GSE66743 and GSE145064 datasets, P<0.05). Immunohistochemical staining results indicated that the expression rate of CDK1 protein in MPNST tissues was 40.31%. Survival analysis results demonstrated that CDK1 expression was associated with poor prognosis. The survival time of MPNST patients with high CDK1 mRNA expression was significantly lower than that of the low expression group ( P<0.05), and the overall survival trend of patients with positive CDK1 protein expression was worse than that of patients with negative CDK1 expression. Additionally, differential analysis of CDK family genes (CDK1-8) revealed that only CDK1 was significantly upregulated in MPNST, NF, and PNF. Increased expression of CDK1 is associated with poor prognosis in MPNST patients. Compared to other CDK family members, CDK1 exhibits a unique expression pattern, suggesting its potential as a therapeutic target for MPNST.

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor A (VEGF-A) expressions in Ethiopian female breast cancer and their association with histopathologic features.

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGF) play important role in breast tumor growth, invasion, metastasis, patient survival and drug resistance. The aim of this study was to evaluate the protein expression status of EGFR and VEGF-A, as well as their association with hormone receptor status and histopathological characteristics in the invasive type of female breast cancer among Ethiopians. The primary breast tumor tissues were obtained from 85 Ethiopian invasive breast cancer cases that underwent modified radical mastectomy (MRM) from June 2014 to June 2015. Their FFPE blocks were analyzed for EGFR and VEGF protein expressions using immunohistochemical techniques. The expressions were also correlated with histopathologic features. Epidermal growth factor receptor over-expression was observed in 22% of the tumor samples. VEGF-A expression was negative in 13.41%, low in 63.41%, moderate in 20.73%, and high in 2.44%. EGFR expression, but not VEGF-A, showed a significant inverse correlation with both estrogen receptor (ER) (P = 0.01) and progesterone receptor (PR) statuses (P = 0.04). EGFR and VEGF expressions did not show significant association with tumor size, grade, lymph node status or age at diagnosis. Epidermal growth factor receptor expression was most likely associated with ER and PR negative tumors. Assessments of multiple molecular markers aid to understand the biological behavior of the disease in Ethiopian population. It might also help to predict which group of patients might get more benefit from the selected treatment strategies and which are not.

Tissue-resident T cells in Clinical Response and Immune-related Adverse Events of Immune Checkpoint Blockade.

T cell surveillance of tissues is spatially organized: circulating memory T cells perform surveillance of secondary lymphoid organs while tissue-resident memory T cells act as sentinels in barrier tissues. In the context of infection, tissue-resident memory T cells survive long term in barrier tissues and are poised to respond to re-encounter of infectious agents. The activity of such tissue-resident T cells is regulated by the PD-1 and CTLA-4 inhibitory receptors targeted by cancer immunotherapies. This review investigates the hypothesis that T cells with a tissue residency program play an important role in both protective anti-tumor immunity and immune-related adverse events (irAEs) of immune checkpoint blockade (ICB). A series of translational studies have demonstrated that a higher density of tissue-resident T cells within tumors is associated with favorable survival outcomes in a diverse range of cancer types. Tissue-resident T cells have also been implicated in clinical response to immune checkpoint blockade, and dynamic tracking of T cell populations in pre- and on-treatment tumor samples demonstrated that T cells with a tissue residency program responded early to ICB. Investigation of colitis and dermatitis as examples of irAEs demonstrated that tissue-resident memory T cells were reactivated at these epithelial sites, resulting in a highly cytotoxic state and secretion of inflammatory cytokines IFNγ and TNFα. It will therefore be important to consider how a tissue residency program can be enhanced to promote T cell-mediated tumor immunity while preventing the development of irAEs.

Multigene Panel Next-Generation Sequencing Techniques in the Management of Patients with Metastatic Colorectal Carcinoma: The Way Forward for Personalized Treatment? A Single-Center Experience

The efficacy and cost-effectiveness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remain uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), and BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in five patients (2.8%) and SMO in four patients (2.2%). Additionally, MAP2K1, CTNNB1, and MYC were mutated in three patients (2.4%). Two mutations (1.1%) were observed in ERBB3, RAF1, MTOR, JAK1, and FGFR2. No significant survival differences were observed based on number of mutations. In total, 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited.

Exploring the impact of sEH inhibition on intestinal cell differentiation and Colon Cancer: Insights from TPPU treatment

Inhibition of soluble epoxide hydrolase (sEH) appears to be promising for the treatment of many diseases. Studies have focused on the beneficial effects of epoxyeicosatrienoic acids (EETs), which are sEH substrates. However, our recent studies have shown that the sEH activity is crucial for the proper intestinal cell differentiation. In this recent study, we investigated the impact of TPPU, an inhibitor of sEH, on the colon cancer cell lines Caco2 and HT-29. We analysed the changes in the expression of the cytoskeletal protein ezrin and the phosphorylated protein kinase p38 (p-p38). Our results showed a decrease in ezrin expression in differentiated cells and an increase in p-p38 expression after TPPU treatment. Immunocytochemical staining revealed a higher staining intensity of p-p38 in the nuclei of HT-29 cells following TPPU treatment. Immunohistochemical staining was performed on human samples of normal colon tissue, grade 2 tumours, and embryonal/foetal tissues. The staining intensity of ezrin in tumours was reduced in the surface area compared to the crypts. Additionally, we observed the translocation of p-p38 expression from the cytoplasm to the nucleus during differentiation. The tumour samples exhibited higher levels of p-p38 in the cytoplasm, similar to normal undifferentiated tissue. To observe the disruption of the cytoskeleton after TPPU treatment, confocal microscopy was used. It was found that β-actin associated with ezrin forms clusters under the plasma membranes. All of these results are significant because sEH inhibitors are being tested in clinical trials, but they could cause an unexpected adverse effects.

Energy Metabolism Behavior and Response to Microenvironmental Factors of the Experimental Cancer Cell Models Differ From That of Actual Human Tumors.

Analysis of the biochemical differences in the energy metabolism among bi-dimensional (2D) and tri-dimensional (3D) cultured cancer cell models and actual human tumors was undertaken. In 2D cancer cells, the oxidative phosphorylation (OxPhos) fluxes range is 2.5-19 nmol O2/min/mg cellular protein. Hypoxia drastically decreased OxPhos flux by 2-3 times in 2D models, similar to what occurs in mature multicellular tumor spheroids (MCTS), a representative 3D cancer cell model. However, mitochondrial protein contents and enzyme activities were significantly different between both models. Moreover, glycolytic fluxes were also significantly different between 2D and MCTS. The glycolytic flux range in 2D models is 1-34 nmol lactate/min/mg cellular protein, whereas in MCTS the range of glycolysis fluxes is 60-80 nmol lactate/min/mg cellular. In addition, sensitivity to anticancer canonical and metabolic drugs was greater in MCTS than in 2D. Actual solid human tumor samples show lower (1.6-4.5 times) OxPhos fluxes compared to normoxic 2D cancer cell cultures. These observations indicate that tridimensional organization provides a unique microenvironment affecting tumor physiology, which has not been so far faithfully reproduced by the 2D environment. Thus, the analysis of the resemblances and differences among cancer cell models undertaken in the present study raises caution on the interpretation of results derived from 2D cultured cancer cells when they are extended to clinical settings. It also raises awareness about detecting which biological and environmental factors are missing in 2D and 3D cancer cell models to be able to reproduce the actual human tumor behavior.

CD74 is a potential biomarker predicting the response to immune checkpoint blockade

BackgroundImmune checkpoint blockade (ICB) has been improving the patient outcome in multiple cancer types. However, not all patients respond to ICB. Biomarkers are needed for selecting appropriate patients to receive ICB. CD74 is an important chaperone that regulates antigen presentation for immune response. However, the relationship between CD74 expression and ICB response remains elusive.MethodsThe unified normalized pan-cancer dataset was downloaded from the UCSC database. Wilcoxon Rank Sum Rank Tests were used to analyze the expression differences between normal and tumor samples in each tumor type. Then, the prognostic value of CD74 was determined using univariable Cox proportional hazards regression analysis. The STRING database was utilized to construct the protein-protein interaction (PPI) network of CD74 and the signal pathways were analyzed as well. The correlation of CD74 expression with immune cells and immune regulating genes was investigated in the TIMER database. The TIDE framework was utilized to evaluate the relationship between CD74 expression and the response to immunotherapy. Moreover, the localization of CD74 in the tumor immune microenvironment was verified using multiplex immunohistochemistry. Clinically annotated samples from 38 patients with esophageal cancer treated with neoadjuvant chemotherapy combined with ICB were analyzed for CD74 expression using immunohistochemistry.ResultsIn this study, we investigated the prognostic and predictive value of CD74 in different types of cancer. Compared with normal tissue, the expression of CD74 was higher in tumor tissue in various cancers. High expression of CD74 was associated with improved patient prognosis in the majority of cancers. CD74 and its interacting proteins were mainly enriched in the immune-related pathways. The expression of CD74 was significantly positively correlated with B cells, CD4 T-cells, CD8 T-cells, neutrophils, macrophages and dendritic cells. TIDE analysis showed that tumors with high CD74 expression may have better responses to immunotherapy and improved patient survival. In patients with esophageal cancer who had received ICB, higher intratumoral CD74 expression was associated with improved response to ICB.ConclusionsThe findings of this study suggest that the high expression of CD74 may be a potential predictive biomarker of response to ICB.

Mapping extrachromosomal DNA amplifications during cancer progression.

To understand the role of extrachromosomal DNA (ecDNA) amplifications in cancer progression, we detected and classified focal amplifications in 8,060 newly diagnosed primary cancers, untreated metastases and heavily pretreated tumors. The ecDNAs were detected at significantly higher frequency in untreated metastatic and pretreated tumors compared to newly diagnosed cancers. Tumors from chemotherapy-pretreated patients showed significantly higher ecDNA frequency compared to untreated cancers. In particular, tubulin inhibition associated with ecDNA increases, suggesting a role for ecDNA in treatment response. In longitudinally matched tumor samples, ecDNAs were more likely to be retained compared to chromosomal amplifications. EcDNAs shared between time points, and ecDNAs in advanced cancers were more likely to harbor localized hypermutation events compared to private ecDNAs and ecDNAs in newly diagnosed tumors. Relatively high variant allele fractions of ecDNA localized hypermutations implicated early ecDNA mutagenesis. Our findings nominate ecDNAs to provide tumors with competitive advantages during cancer progression and metastasis.

Flower-Shaped Plasma Cells in Multiple Myeloma with Morphological Heterogeneity.

Flower-shaped nuclei in plasma cells are rare in multiple myeloma. We report on an 88-year-old male who presented with a mass lesion in the clavicular region. A biopsy of the mass revealed an increase in mature plasma cells with round nuclei. In contrast, a bone marrow examination showed increased plasma cells with flower-shaped nuclei. The patient tested negative for human T-lymphotropic virus type-1 and was diagnosed with multiple myeloma. While multiple myeloma is known for intra-tumor heterogeneity, reports of morphological heterogeneity based on the site of tumor sampling are limited. In this case, the presence of plasma cells with flower-shaped nuclei enabled the identification of site-dependent morphological tumor heterogeneity.

Prediction of homologous recombination deficiency identifies colorectal tumors sensitive to PARP inhibition

The synthetic lethal effect observed with the use of PARP inhibitors (PARPi) with tumors characterized by the loss of key players in the homologous recombination (HR) pathway, commonly referred to as “BRCAness”, is maintaining high interest in oncology. While BRCAness is a well-established feature in breast, ovarian, prostate, and pancreatic carcinomas, our recent findings indicate that up to 15% of colorectal cancers (CRC) also harbor defects in the HR pathway, presenting promising opportunities for innovative therapeutic strategies in CRC patients. We developed a new tool called HRDirect, which builds upon the HRDetect algorithm and is able to predict HR deficiency (HRD) from reference-free tumor samples. We validated HRDirect using matched breast cancer and CRC patient samples. Subsequently, we assessed its efficacy in predicting response to the PARP inhibitor olaparib by comparing it with two other commercial assays: AmoyDx HRD by Amoy Diagnostics and the TruSight Oncology 500 HRD (TSO500-HRD) panel by Illumina NGS technology. While all three approaches successfully identified the most PARPi-sensitive CRC models, HRDirect demonstrated superior precision in distinguishing resistant models compared to AmoyDX and TSO500-HRD, which exhibited overlapping scores between sensitive and resistant cells. Furthermore, we propose integrating HRDirect scoring with ATM and RAD51C immunohistochemical analysis as part of our “composite biomarker approach” to enhance the identification of HRD tumors, with an immediate translational and clinical impact for CRC personalized treatment.

Differential regulation of expression of the protein kinases DYRK1A and DYRK1B in cancer cells

The protein kinases DYRK1A and DYRK1B are pivotal regulators of cell cycle progression by promoting cell cycle exit into quiescence. DYRK1B appears to play a more important role in cancer cell quiescence than DYRK1A, as evidenced by its overexpression or copy number variations in human tumour samples. Nonetheless, the stimuli driving DYRK1B upregulation and the potential divergence in expression patterns between DYRK1A and DYRK1B remain largely elusive. In the present study, we scrutinized the regulatory pathways modulating DYRK1B expression relative to DYRK1A in PANC-1 and A549 cancer cell lines across varying conditions. Serum deprivation, pharmacological mTOR inhibition and increased cell density resulted in the differential upregulation of DYRK1B compared to DYRK1A. We then aimed to assess the role of protein kinases MST1 and MST2, which are key transmitters of cell density dependent effects. Unexpectedly, exposure to the MST1/2 inhibitor XMU-MP-1 resulted in increased DYRK1B levels in A549 cells. Further investigation into the off-target effects of XMU-MP-1 unveiled the inhibition of Aurora kinases (AURKA and AURKB) as a potential causative factor. Consistently, AURK inhibitors VX-680 (tozasertib), MLN8237 (alisertib), AZD1152-HQPA (barasertib) resulted in the upregulation of DYRK1B expression in A549 cells. In summary, our findings indicate that the expression of DYRK1A and DYRK1B is differentially regulated in cancer cells and reveal that the kinase inhibitor XMU-MP-1 increases DYRK1B expression likely through off target inhibition of Aurora kinases.

Structural alterations of the EGFR gene in glioblastoma samples as a prognostic factor and molecular target for therapy

Introduction. Epidermal growth factor receptor (EGFR) is a transmembrane protein of the receptor tyrosine kinase family that is activated in various cancers (non-small cell lung cancer, colorectal cancer, head and neck tumors). In glial brain tumors, increased EGFR expression levels are characteristic of the most aggressive subtype, glioblastoma. Frequent structural changes of EGFR in glioblastoma are amplification of the chromosome region where the EGFR gene is located, point mutations, as well as deletion of exons 2–7 of the EGFR gene leading to the formation of EGFRvIII transcript.Aim. To determine structural changes of the EGFR gene (point mutations and amplification of the EGFR gene, EGFRvIII transcript) in tumor samples using different methods and to evaluate their potential clinical significance.Materials and methods. The study included 75 patients with brain gliomas (70 of them glioblastoma) aged 34 to 78 years (mean age 56 years). DNA and RNA isolation was performed from fresh frozen tumor tissue, as well as from peripheral blood leukocytes. EGFR gene mutations were determined by next-generation sequencing (NGS), and β allele frequency (BAF) comparative analysis (normal-tumor) was performed to determine the copy number of chromosome 7 regions. Quantitative polymerase chain reaction was used to confirm the EGFR gene amplification in tumor samples, and reverse transcription-PCR was used to detect EGFRvIII variant.Results. The NGS method revealed 11/70 (16 %) mutations in coding regions of EGFR gene in glioblastoma samples, the EGFR gene amplification was detected in 26/70 (37 %) cases; no structural changes of the EGFR gene were detected in 5 glioma samples (astrocytoma, oligodendroglioma). All cases of EGFR gene amplification detected by NGS were confirmed by quantitative polymerase chain reaction. To search for EGFRvIII transcript, 31 tumor RNA samples were examined, of which EGFR amplification was present in 12 samples. EGFRvIII transcript was detected only in samples with EGFR gene amplification – 4/12 (33 %). To assess the clinical significance of structural gene alterations, the frequency of occurrence in primary and recurrent glioblastoma samples was compared.Conclusion. The NGS method allows to detect both point mutations and amplification of the EGFR gene. The EGFR gene amplification was associated with EGFRvIII mutation in 33 % of cases. No statistically significant differences in the frequency of structural changes in the EGFR gene between primary and relapsed glioblastomas were found.

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Drivers in Serrated Pathway-Driven Colorectal Cancer Progression

Serrated lesions are common precancerous pathways in colorectal cancer (CRC), but the process by which they progress to malignancy remains unclear. We aimed to elucidate this progression through a single-cell RNA landscape. We conducted single-cell RNA sequencing on three normal colonic tissues and fifteen SLs (including HPs, SSLs, SSLD, and TSAs) and integrated these data with datasets containing tumor samples. We identified three invasive malignant epithelial cell subtypes related to CRC progression: SLC1, SLC2, and tumor cell. SLC1, specific to SSLs, is involved in cell proliferation and shows a continuum of malignancy in gene expression. TSA-specific SLC2 exhibited FOXQ1 upregulation and active EMT, indicating invasiveness. The trajectory analysis showed that HPs do not progress to cancer, and different SL types are linked to the MSI status of advanced CRCs. We validated molecular drivers in premalignant lesions and later carcinogenesis. In the tumor microenvironment, CAF and pre-CAF fibroblast subtypes associated with progression were identified. During the premalignant stage, SLC1 triggered CD8+ T cell responses, while at the advanced stage, CAFs promoted tumor invasion and metastasis via FN1-CD44, influencing tumor progression and the treatment response. Our findings highlight transcriptional changes across serrated pathway stages, aiding in early CRC diagnosis and treatment.